The research goal of the ROO phase of this Pathway to Independence Award is to develop a general purpose multiplexing fluorescence microscopic platform with single monomer sensitivity. The instrument will perform lifetime and radiometric measurements on excitation and emission simultaneously, which allows quantitative measurements on multi-color FOrster Resoiiant Energy Transfer (FRET). In the K99 phase of the award, we worked on developing a high-speed Simultaneous Fluorescence Lifetime Excitation Emission (SFLEE) spectrometer. In the ROO phase, we will improve the sensitivity of the SFLEE technique, and apply the technique to multi-color FRET investigations. Fast lifetime and intensity excitation-emission matrices obtained by the SLFEE instrument will allow real time measurements of FOrster Resonant Energy Transfer between more than three fluorescent labels. To demonstrate the instrument's capability, we perform quantitative four-color FRET on single DNA Holliday junction. Specifically, we will study structure dynamics of single DNA Holliday junctions undergoing global confirmation switching and branch migration. The long term objective of this research is to provide a high-throughput multiplexed imaging tool for both fundamental and biomedical research.